P1360: CLINICAL TRANSLATION OF A NOVEL, POTENT AND WELL-CHARACTERIZED INDUCED REGULATORY T-CELL PRODUCT AGAINST GRAFT VERSUS HOST DISEASE
Authors: Memnon Lysandrou, Dionysia Kefala, Panayiota Christofi, Penelope Georgia Papagianni, Antonis Mingos, Chryso Pieridou, Elisavet Vlachonikola, Maria Kyriakou, Alexandra Lydia Chatzidaniil, Nikolaos Savvopoulos, Anastasia Papadopoulou, Anastasia Chatzidimitriou, Paul Costeas, Evangelia Yannaki, Alexandros Spyridonidis
Journal: HemaSphere (2023)

Topic: 24. Gene therapy, cellular immunotherapy and vaccination - Biology & Translational Research Background: Immunotherapy with regulatory T cells (Tregs) stands as an alternative treatment for Graft-versus-Host Disease (GvHD), a major complication of allogeneic hematopoietic cell transplantation (allo-HCT). We have previously reported that HLA-G expression, known to promote feto-maternal tolerance, is induced by pharmacological hypomethylation of T cells, generating induced HLA-G+ Tregs with suppressive properties. Aims: To generate a fully characterized, safe and efficacious clinical-scale and GMP-grade product enriched in HLAG+ T cells (iG-Tregs), and ultimately evaluate it in a phase I/II trial in patients undergoing HLA-matched sibling allo-HCT against GvHD. Methods: Peripheral blood mononuclear cells from healthy donors were enriched for T-cells by adherence-based monocyte depletion, activated with anti-CD3/CD28 antibodies and treated for 3 days with decitabine. The safety and efficacy profile of the iG-Tregs product was assessed in vitro and in vivo. Polyclonal expansion of iG-Tregs was assessed by Immune-seq (Illumina). Sorted HLAG+ cells were analysed by bulk (Illumina) and single-cell RNAseq (10X Genomics). Results: iG-Tregs (n=22) contained 91.3% CD3+ cells (median-Md) with 16.6% CD3+HLAG+ cells (2.4-28%) which was higher than PBS-Treated cells (controls) (1.4%, p<0.0001). A predominant CD4+ fraction was observed versus controls (67.7% vs 57%). Immunophenotyping of iG-Tregs revealed the expression of TIM3 and LAG3 inhibitory receptors stemming mostly from HLAG+ cells. Except for IL13 (p=0.05), no other Th1/2/17 cytokines were increased in iG-Tregs supernatants, a finding which was validated by FACS. Immunoprofiling of TRBV-TRBD-TRBJ gene rearrangements revealed that the extremely diverse and polyclonal TR gene repertoire of controls was retained in iG-Tregs, while treatment drove to repertoire renewal as only 4% (Md, 2%-4.7%) of the total identified T cell clones were shared amongst controls and HLAG+ population’s repertoire. RNAseq revealed HLAG+ cells to posses a distinct and homogenous transcriptional profile and upregulate the myeloid suppressive genes IDO-1, CCL17 and CCL22 compared to their HLAG- counterparts, while single-cell RNAseq uncovered underlying regulatory signatures. Moreover, iG-Tregs demonstrated a favourable safety profile over controls in vitro, showing diminished alloreactivity against allogeneic targets (1.8%vs49% lysis) and their hyporesponsiveness after restimulation via reduction of intracellular pro-inflammatory cytokines (IL2/IFNγ/IL17a/TNFα). The diminished alloreactivity of iG-Tregs over controls was confirmed in vivo after infusion in NSG mice; 6/9(67%) iG-Tregs-treated mice survived until sacrifice whereas all 7 mice infused with controls developed GvHD from day 21 and succumbed by day 35 (Md). iG-Tregs’ ability in preventing GvHD was tested by their co-infusion with T-cells; while T-cell infusion resulted in lethal GvHD by day 36 in all 3 control mice, the co-administration of iG-Tregs delayed (6/8 mice died by day 66) or even prevented (2/8) GvHD onset with a second infusion increasing survival to 40%. Respectively, GMP-iG-Tregs were comparable with preclinical products regarding immunophenotype, immunoprofile, and absence of alloreactivity. Summary/Conclusion: iG-Tregs are enriched in regulatory HLAG+ cells and can be robustly and reproducibly generated through a short and GMP-compatible protocol. iG-Tregs constitute a well-characterized product with the safety profile required for clinical translation, leading to the approval and initiation of a phase I-II clinical trial for GvHD (EUDRACT 2021-006367-26). Keywords: Hypomethylating agents, Regulatory T cell, Graft-versus-host disease (GVHD), Allogeneic hematopoietic stem cell transplant